Our long-term goal is to engineer functionally competent cartilage for replacing damaged or diseased cartilage. While it is known that engineering a functionally competent cartilage depends on the mechanical environment, choosing the appropriate loading environment has proven challenging. This observation is largely in part due to the fact that the biomechanical cues sensed by the cells will be dictated by the mechanical structure and chemistry of the scaffold and will be dynamic in time as the scaffold degrades and neotissue develops. To overcome these challenges, the global hypothesis for this research is that a dynamic culture environment that detects and responds to changes in a tissue-engineered scaffold improves the quality of the engineered cartilage. Central to our hypothesis is a novel dynamic compressive bioreactor recently designed, constructed and validated with collaborators at NIST, which is equipped with online, nondestructive measurement capabilities comprised of individual load cells for assessing mechanical properties and an ultrasonic transducer coupled with a video microscope for assessing development and quality of the engineered tissue. To test the global hypothesis, the specific aims of the project are to: 1. Design a dual enzyme degrading polyethylene glycol (PEG) hydrogel with cell-mediated local degradation and 'on demand' bulk degradation capabilities. This aim tests the hypothesis that a hydrogel with crosslinks containing oligopeptides that are degraded by cells, leading to local degradation (critical for local matrix elaboration without sacrificing mechanical integrity) and polycaprolactone that is degraded 'on demand' by exogenous delivery of lipase, leading to bulk degradation (critical for macroscopic tissue development) yields improved engineered cartilage. 2. Develop and validate a dynamically responsive 'smart' bioreactor using a heuristic control loop to modify the biochemical and mechanical environment in real time. This aim tests the hypothesis that real time changes to bulk degradation and mechanical loading in response to the developing tissue will lead to improved engineered cartilage. We will achieve this aim by incorporating a fuzzy controller with a set of heuristic control actions into our current bioreactor where the output variables, the quality index estimated from ultrasound and mechanical properties, will be related to input variables that include strain amplitude, duty cycle, and enzyme addition. At the completion of this exploratory research, we expect to have developed i) a new class of dual enzyme degrading hydrogels based on cross-linked polyethylene glycol where degradation is more closely matched spatially and temporally to tissue growth and elaboration and ii) a dynamically responsive 'smart' bioreactor that is capable of detecting and responding in accord with tissue growth. We also expect to have answered the fundamental question; does a dynamically responsive culture environment lead to improved tissue elaboration and functional properties over a constant culture environment? It is anticipated that such a bioreactor would enable facile adaption to engineering cartilage from multiple cell sources (donor, age, species), which inherently have different dynamics and timescales for tissue development, and can readily be adapted to other scaffold types. Findings from this grant will lay the foundation for seeking competitively a NIH R01 and to pursue their (pre)clinical utility.